1. Field of the Invention
The present invention relates to a method for the visual indication of cholesterol on the surface of skin utilizing affino-enzymatic compounds based on a cholesterol affinant binding agent and a visualizing agent, and methods for producing such compounds.
By virtue of its simplicity and non-invasive nature, the proposed invention is an ideal tool for mass examinations to identify individuals with advanced atherosclerosis as well as those at risk of developing this disease and other diseases which result in or are attributable to high blood cholesterol levels.
2. Description of Related Art
Many studies in recent years have shown that atherosclerosis and its complications such as heart attacks, strokes and gangrenes, are major causes of mortality in all countries of the world.
Cost effective prevention of atherosclerosis in a population require the identification of the high-risk group of individuals followed by their differentiated treatment and change of life style. The difficulty lies in deciding which individuals belong to the high-risk group.
Existing methods for diagnosing atherosclerosis are based on measuring general or total cholesterol content of venous blood plasma. (Consensus Conference on Lowering Blood Cholesterol to Prevent Heart Disease, JAMA, 1985, 253, pg. 2080; The Lipid Research Clinics Population Studies Data Book, publication 80-152, Bethesda, Md., National Institute of Health, 1980, vol. I; Lipid Research Clinics Program, JAMA, 1984, 251, pg. 351-364). In some countries, a cholesterol content of over 240 mg/dL is considered sufficient to include a patient into the high-risk group. Unfortunately, total cholesterol levels do not accurately predict a patient's risk level.
A more precise diagnosis can be made by analyzing blood plasma lipoproteins and determining the atherogeneity index which is determined by dividing the difference between the total cholesterol and the high-density lipoprotein (HDL) cholesterol, by the high-density lipoprotein cholesterol: EQU Atherogeneity Index=Ch (total)-ChH(HDL)/Ch(HDL)
This index is considered superior for predicting the degree of atherosclerosis within the arteries. A patient is considered to belong to the high-risk group when his atherogeneity index is more than 3, and to the atherosclerosis group, when this index is over 5.6 (Klimov A. N. "Phenotyping of Lipoproteins", Methodological Recommendations of the USSR Ministry of Health, M., Medicine, 1975; Goldfourt v., Koltsman E., Neufeld H. N., total and High Density Lipoprotein Cholesterol in the Serum and Risk of Mortality. British Medical Journal, 1985, 290, pg. 1239-1243).
Use of these methods necessitates blood taking which is uncomfortable and poses a risk of infection. Detailed analysis of plasma lipoproteins and cholesterol is complicated and expensive. Moreover, recent studies showed that the blood plasma does not fully reflect the process of cholesterol accumulation which is typical for the arterial wall and other bradytrophic tissues. In one case out of three, quantification of total cholesterol and even a complete phenotyping do not correlate with the severity of atherosclerosis (See: Myasnikov A. L., "Hypertensive Disease", 1965, M., Medicine, pg. 300).
It has been shown that tissue cholesterol plays a leading role in development of atherosclerosis. Tissues referred to collectively as bradytrophic tissues, have been identified which accumulate cholesterol in the same way as the arterial wall. These studies have demonstrated a close correlation between cholesterol content in the arterial wall and the skin.
This correlation between cholesterol content in the arterial wall and skin was proved through direct measurement of cholesterol in skin samples. Specimens were frozen in liquid nitrogen and lyophilized; cholesterol was extracted with Folch reagent and quantified using traditional chemical or biochemical methods. (See: Nikitin Y. P., Gordienko I. A., Dolgov A. V., Filimonova T. A. "Cholesterol content in the skin and its correlation with lipid quotient in the serum in normals and in patients with ischemic cardiac disease", Cardiology, 1987, II, No. 10, P.48-51; Bouisson H., de Graeve, Solera M. L. et al. Ann. Biol. Clin., 1982, vol. 40, p.361-407). Unfortunately, this method is too complicated and painful to be useful for population screening.
U.S. Pat. No. 4,458,686 describes a method of quantifying the amount of glucose and ethanol localized in the blood directly under the skin or on its surface. This patent also discloses that it is possible to quantify the amount of cholesterol using cholesterol oxidase. The method is based on stoichiometric changes of oxygen concentration using redox enzymes, mostly oxidoreductases, specific to the substrate in question. According to this method, oxygen concentration changes are measured electrochemically; for instance, via polarography with the use of special equipment and a specially designed electrode. Such sophisticated instrumentation necessitates the services of highly skilled personnel for making diagnoses and inevitably and unfortunately limits the usefulness of this method for population screening.
PCT Application US 84/00888 proposes a detection-visualization complex where the binding and visualizing elements are linked directly or through a bridging agent. The complex is intended to detect low quantities of target molecules, including lipids in biological tissues. However, this method of detecting lipids can be used only in laboratory conditions and requires taking biological fluid or tissue samples from a patient. Again, the method is painful, multi-staged and too complicated to be useful for population screening.
Correlation between cholesterol content in the skin and severity of atherosclerotic vascular lesions can be obtained through standard laboratory analysis of skin specimens. In addition to the pain involved in taking a skin sample, this method has other disadvantages because 1 mm thick skin specimens include various skin layers horny layer (average thickness 0.1 mm) and connective tissue (derma proper) represented by two layers--papillary and retinal. Both layers have a good blood supply and, consequently, specimens include vessels and blood; they may also contain perspiratory and sebaceous glands and their secretions. The subcutaneous fat is located directly under the derma and may also get into specimens, i.e. heterogeneity of specimens may pervert the data on cholesterol accumulated in the skin. From this point of view, a method which can quantify the cholesterol content in the horny layer of the derma on the skin surface would be most accurate and preferred.